Modulating Human Cas9-Specific Host Immune Response

ABSTRACT

Provided herein are methods and compositions for reducing an undesirable T cell immune response in human patients prior to and/or during gene therapy using CRISPR/Cas9-based genetic modulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Serial Nos. 62/535,516, filed Jul. 21, 2017, and 62/556,061,filed Sep. 8, 2017, each of which is incorporated by reference herein asif set forth in its entirety.

BACKGROUND

The CRISPR/Cas9 gene-editing tool is currently in clinical trials as theexcitement about its therapeutic potential is exponentially growing.Being a bacterial protein, Cas9 is likely to trigger cellular andhumoral immune reaction in humans, as has been recently demonstrated innaïve mice. The potential consequences of this immune response includeneutralization of the gene product; destruction of the cells expressingit leading to loss of therapeutic activity or tissue destruction; memoryeffect that prevents re-administration; and violent innate inflammatoryresponse.

Cas9-specific T cell activation and proliferation were confirmed in anexpanded CD45+ cell population following in vivo expression of Cas9delivered by an AAV vector or DNA electroporation in mice. These datahighlight the need to characterize the immunogenicity of Cas9 in humansas this gene-editing technology is moving to the clinic. Accordingly,there remains a need for strategies for decreasing the immunogenicity ofCRISPR/Cas9 system components and improving the safety of CRISPR-basedgene therapies for human subjects.

SUMMARY

Provided herein are methods and compositions for decreasing an undesiredT cell immune response in human subjects undergoing gene therapy using aCRISPR/Cas9 system.

In a first aspect, provided herein is a method of identifying andtreating a subject at risk of having a Cas9 antigen-specific CD8+ T cellimmune response. The method can comprise or consist essentially of (a)detecting one or more immunodominant Cas9 epitopes in a biologicalsample obtained from the subject, wherein the detection of the one ormore immunodominant Cas9 epitopes identifies the subject as havingpre-existing immunity to Cas9; and (b) treating the subject identifiedin (a) with CRISPR/Cas9-based gene therapy, wherein treating comprisesintroducing into a cell from the identified subject an engineered,non-naturally occurring Type II CRISPR-Cas system comprising amultifunctional Cas9 protein and at least one guide RNA that targets andhybridizes to a target sequence of a DNA molecule in a cell, wherein theDNA molecule encodes and the cell expresses at least one gene product,and wherein the Cas9 protein comprises a mutation selected from thegroup consisting of L241G, L616G, and L241G/L616G with reference to theposition numbering of a Streptococcus pyogenes Cas9 protein (SEQ IDNO:1), whereby expression of the at least one gene product is alteredand a disease associated with the gene product is treated. Theintroducing step can be performed ex vivo or in vivo.

In another aspect, provided herein is a method of reducing an undesiredCas9-specific CD8+ T cell immune response in a subject who will receiveCRISPR/Cas9-based gene therapy. The method can comprise or consistessentially of the method introducing into a cell from a subjectidentified as having pre-existing immunity to Cas9 an engineered,programmable, non-naturally occurring Type II CRISPR-Cas systemcomprising a multifunctional Cas9 protein and at least one guide RNAthat targets and hybridizes to a target sequence of a DNA molecule in acell, wherein the DNA molecule encodes and the cell expresses at leastone gene product, and wherein the Cas9 protein comprises a mutationselected from the group consisting of L241G, L616G, and L241G/L616G asnumbered relative to SEQ ID NO:1, whereby expression of the at least onegene product is altered and whereby a Cas9-specific CD8+ T cell immuneresponse is reduced relative to that produced by a cell comprising anaturally occurring Cas9 or an engineered, programmable, non-naturallyoccurring Type II CRISPR-Cas system wherein the Cas9 protein does notcomprise the mutation. The introducing step can be performed ex vivo orin vivo.

In another aspect, provided herein is a variant Cas9 protein encoded bythe amino acid sequence of SEQ ID NO:2, a variant Cas9 protein encodedby the amino acid sequence of SEQ ID NO:3, and a variant Cas9 proteinencoded by the amino acid sequence of SEQ ID NO:4.

In a further aspect, provided herein is an isolated polynucleotideencoding a variant Cas9 polypeptide, a vector comprising such apolynucleotide, and a host cell comprising such a vector.

In another aspect, provided herein is a method of making a variant of aCas9 polypeptide shown in SEQ ID NO:1. The method can comprise orconsist essentially of using a polynucleotide mutagenesis procedure togenerate a population of mutants of the Cas9 polynucleotide shown in SEQID NO:5, wherein the population of mutant Cas9 polynucleotides encodesCas9 polypeptide variants having at least one amino acid substitutionselected from the group consisting of L241G and L616G as numberedrelative to SEQ ID NO:1; and expressing a population of Cas9 polypeptidevariants encoded by the population of Cas9 polynucleotide mutants; sothat a variant of a Cas9 polypeptide shown in SEQ ID NO:1 is made. Themethod can further comprise screening one or more members of thepopulation of Cas9 polypeptide variants so as to identify a variant thatexhibits a decreased immunogenicity as compared to the Cas9 polypeptideshown in SEQ ID NO:1 but retains cleavage and/or binding activityrelative to the activity of a Cas9 polypeptide without the at least oneamino acid substitution.

In a further aspect, provided herein is a variant Cas9 protein made bythe method described above, wherein the variant Cas9 protein is encodedby an amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3, and SEQ ID NO:4.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood and features, aspects,and advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIG. 1 is a schematic illustrating a workflow for selecting immunogenicepitopes.

FIG. 2 demonstrates detection of specific serum antibodies (Abs) againstS. pyogenes lysate in 49.0% (above the dotted line) of 143 healthycontrols (left). The subset shown in black circles was screened for Absagainst synthetic wild-type Cas9 protein (right), of which 36.6% (21.0%of total samples screened) were positive (above the dashed line). Linesrepresent median and interquartile range. The mean Relative Light Units(RLU) of positive sera was comparable to the mean RLU detected againstthe EBNA-1 protein for sera with known EBNA-1 seropositivity.

FIG. 3 demonstrates the detection of pre-existing immune response toSpCas9 in healthy donors and identification of two immunodominant T-cellepitopes. The top 5 predicted SpCas9 T cell epitopes and their predictedSb and Si scores and ranking (based on the Sb. Si value). These top 5peptides include the identified immunodominant (α and β; gray) andsubdominant (γ and δ) epitopes that were shown to be immunogenic byIFN-γ ELISpot. The plot shows Sb and Si of predicted HLA-A*02:01epitopes for the SpCas9 protein. Red dots represent the immunodominant(α and β) and subdominant (γ and δ) epitopes.

FIG. 4 presents scatter dot plots (median with interquartile range) ofIFN-γ ELISpot measurements of T cell reactivity of 12 healthy donors to38 predicted epitopes grouped in 10 pools, CEF (positive control), andDMSO (negative control). Dots represent the means of triplicate wellsfor each sample and 2 independent replicates.

FIG. 5 is three-dimensional (3D) representation of the structure ofSpCas9 protein, showing the location of the identified immunodominantepitopes α (previously identified as epitope 85) and β (previouslyidentified as epitope 94).

FIG. 6 shows IFN-γ ELISpot reactivity of healthy donor T cells (n=12) toepitopes across the different domains of the Cas9 protein. Epitopes αand β were the most reactive. Results represent the means of triplicatewells for each sample and 2 independent replicates.

FIGS. 7A-7J demonstrate pCas9 immunodominant epitope-specific CD8+ Tcell recognition is abolished after anchor residue mutation while themutated SpCas9 protein retains its function and specificity. (A) Epitopeβ-specific CD8+ T cell response detected using β-specific pentamer inPBMCs stimulated with peptide β-pulsed antigen presenting cells. (B) Thepercentage of CD8+β-pentamer+ T cells was reduced to 0.3% when APCs werepulsed with the mutated peptide β2. (C) Sequences of epitopes α and βbefore and after mutation of the anchor (2nd and/or 9th) residues. Sb,normalized binding score; Si, normalized immunogenicity score. (D) IFN-γELISpot assay in triplicate wells comparing T cell reactivity to wildtype or mutated epitopes α and β. These results are representative of 12donors and two independent replicates. (E) Schematic of the experimentassessing mutagenesis capacity of Cas9-β2. Cells were transfected witheither WT-Cas9, Cas9-β2, or an empty plasmid as well as 20 nt gRNAtargeting EMX-1 locus. 72 hr after the transfection, percent cleavagewas assessed by DNA extraction and illumine sequencing. (F) Percentageof indel formation in EMX-1 locus. Each individual dot represents anindividual transfection. (G) Schematic of the experiment assessing gRNAbinding, DNA targeting and transcriptional modulation with Cas9-β2.Cells were transfected with either WT-Cas9, Cas9-β2, or an empty plasmidas well as 14 nt gRNA targeting TTN or MIAT in the presence ofMS2-P65-HSF1 (transcriptional modulation). 72 hr after the transfection,mRNA was assessed by qRT-PCR. (H and I) Shown is the mRNA level relativeto an untransfected control experiment. Each individual dot representsan individual transfection. (J) Mean expression levels of 24,078 proteincoding and non-coding RNA genes for WT-Cas9 and Cas9-β2 (each induplicate) are shown. For visualization purposes, the values weretransformed to a log 2(CPM+1) scale. MIAT, the gRNA target gene, ishighlighted in red, and R denotes Pearson correlation coefficientbetween two groups.

FIGS. 8A-8B demonstrate reduced T cell response to epitopes α and βafter mutation of the anchor residues. (A and B) IFN-γ ELISpot for 12healthy donor PBMCs stimulated with wild type or mutated peptide α (A)or β (B). The average reduction was 8 fold from α to α29 and 25 foldfrom β to β29 (p<0.047). Data represent mean of triplicates and twoindependent replicates +/−SEM. Two-tailed p value is calculated forpaired t tests. A representative ELISPOT image is represented in theFIG. 7D.

FIGS. 9A-9B. (A)(left) Schematic of the experiment assessing Cas9-β2cleavage capacity at a synthetic promoter. Cells were transfected witheither WT-Cas9, Cas9-β2, or an empty plasmid as well as 20 nt gRNAtargeting a synthetic CRISPR promoter that harbors two gRNA target sitesflanking a mini-CMV promoter. The targeting and cleavage at the promotershould disrupt the promoter and decrease EYFP expression. (A)(right) Bargraphs show mean+/−SD of geometric mean of EYFP expression 48 hoursafter the transfection in cells expressing >2×10² A.U of a transfectionmarker measured by flow cytometry (n=2 individual transfectionsrepresented by individual dots). (B)(left) Schematic of the experimentassessing Cas9-β2 transcriptional activation capacity at a syntheticpromoter. Cells were transfected with either WTCas9, Cas9-β2 or an emptyplasmid as well as aptamer binding transcriptional activation domains,and 14 nt gRNA targeting a synthetic CRISPR promoter that harborsmultiple target sites upstream of a mini-CMV promoter. The targeting atthe promoter should enable iRFP expression. (B)(right) Bar graph showsmean+/−SD of geometric mean of iRFP expression 48 hours after thetransfection in cells expressing >2×10² A.U of a transfection markermeasured by flow cytometry (n=3 individual transfections represented byindividual dots. n=2 for No Cas9 group).

FIGS. 10A-10F. (A) Analysis of mutagenesis capacity of Cas9-α2 ascompared to WT Cas9 in a synthetic promoter. Bar graph shows mean+/−SDof geometric mean of EYFP expression 48 hr after transfection in cellsexpressing >2×10² A.U of a transfection marker measured by flowcytometry (n=2 individual transfections, represented by individualdots). (B) Mutagenesis in endogenous EMX-1 locus. Percentage of indelformation in EMX-1 locus. Dots represent three individual transfections.(C) Transcriptional modulation by Cas9-α2 at a synthetic promoter. Bargraph is mean+/−SD of geometric mean of iRFP expression 48 hr after thetransfection in cells expressing >2×10² A.U of a transfection markermeasured by flow cytometry (n=3 individual transfections, n=2 for noCas9 group represented by dots). (D, E) Shown is mRNA level relative toan untransfected control experiment. Each individual dot representindividual transfections. WT-Cas9 data and no Cas9 data for panels A andC are also presented in FIGS. 9A-9B. For B, D, and E, WT-Cas9 data andno Cas9 data are also presented in FIGS. 7A-7J. (F) Activated CD8+CD137+T cells detected in PBMCs stimulated with peptide α was reduced in PBMCsstimulated with peptide α2.

FIGS. 11A-11B. (A) Representative flow cytometry gating for analysis ofCas9 function on synthetic promoters. Cells are gated based on Forward(FSC) and Side Scatter (SSCs). Transfected population then was gatedbased on expression of EBFP (BV421-A) more than 2×10{circumflex over( )}2. The geometric mean of the output (EYFP) or iRFP was determined inthis population. The BB515-A graph (far right) shows EYFP expression incells expressing >2×10² A.U of the transfection marker (BV421). (B) Flowcytometry gating for analysis of Cas9 pentamer+CD8+T lymphocytes. Cellswere gated based on FSC and SSC and negatively gated onCD4/CD14/CD19/CD56. The CD8-, CD8+, and Cas9 pentamer+ population areshown (bottom right).

FIG. 12 is a table presenting Cas9 HLA-A*02:01 epitopes (SEQ IDNOs:24-61 for epitopes ranked 1-38, respectively) predicted using theprediction model described herein and ranked according to theirS_(i)-S_(b) score.

FIG. 13 is an amino acid sequence encoding CRISPR-associatedendonuclease Cas9/Csn1 of Streptococcus pyogenes (UniProtKB Q99ZW2) (SEQID NO:1).

FIG. 14 is a nucleotide sequence encoding CRISPR-associated endonucleaseCas9 of Streptococcus pyogenes (NCBI Reference Sequence: NC 002737.2)(SEQ ID NO:5).

While the present invention is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments isnot intended to limit the invention to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

DETAILED DESCRIPTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though set forth in their entirety in the presentapplication.

Although the following description refers to certain aspects orembodiments, such aspects or embodiments are illustrative andnon-exhaustive in nature. Having reviewed the present disclosure,persons of ordinary skill in the art will readily recognize andappreciate that numerous other possible variations or alternativeconfigurations or aspects are possible and were contemplated within thescope of the present disclosure.

Provided herein are compositions, methods, and systems based at least inpart on the inventors' identification of immunodominant T cell epitopesof Streptococcus pyogenes Cas9 (SpCas9).

In a first aspect, provided herein is a method of making a geneticallymodified Cas protein and variant Cas proteins made by such methods.Modifications to a Cas9 protein sequence are made based onimmunodominant T cell epitopes of Cas proteins, including wild-type andmodified (variant) versions of Cas9. In some embodiments, a Cas9 proteinhas been genetically modified to remove immunogenically dominantepitopes (also known as “immunodominant epitopes”) associated withsuboptimal results in clinical gene therapy. As used herein, the term“immunodominant” refers to an epitope capable of stimulating an immuneresponse over other potential epitopes contained within a protein ororganism. Deletion or mutation of immunodominant epitopes canpotentially decrease the risk of the potentially disruptive immuneresponse in individuals before or during receiving CRISPR/Cas treatment.This method specifically modifies a Cas protein to reduce the risk ofthe potentially disruptive immune response in individuals (regardless oftheir HLA type) while preserving its function. In this manner,immunogenic epitopes will be silenced to generate a CRISPR/Cas tool thatinduces minimal host immune response. As used herein, the term“epitope,” also known as an immunogenic epitope or antigenicdeterminant, refers to the set of amino acid residues that is involvedin recognition by a particular immunoglobulin, or in the context of Tcells, those residues necessary for recognition by T cell receptorproteins and/or Major Histocompatibility Complex (MHC) receptors. In animmune system setting, in vitro or in vivo, an epitope is the collectivefeatures of a molecule, such as primary, secondary and tertiary peptidestructure, and charge, that together form a site recognized by animmunoglobulin, T cell receptor, or Human Leukocyte Antigen (HLA)molecule.

Immunodominance is the observation that in spite of a large number ofpossible epitopes (antigen fragments) in an antigen, the immune systemfocuses its response on a limited number of epitopes and can be orderedas a reproducible hierarchy (Sercarz et al. 1993). Immunodominance holdstrue for immune responses to artificial antigens, human virusesincluding influenza and vaccinia, and intracellular bacteria (Chen W S1994, Belze G T et al. 2000, Chen W 2000, Tscharke D C 2005). As usedherein, the term “dominant antigen” or “dominant epitope” (also referredto herein as an “immunodominant epitope”) refers to an antigen orepitope that evokes a strong tolerance or immune response, which may becharacterized by the presence of T cells specific for that antigen orepitope in an amount greater than about 70% of the total number ofresponding T cells. As used herein, the term “subdominant antigen” or“subdominant epitope” refers to an antigen or epitope that evokes aweaker tolerance or immune response than that of a dominant antigen orepitope.

As used herein, the term “immune response” refers to a response of acell of the immune system, such as a B cell, T cell, or monocyte, to astimulus. In one embodiment, the response is specific for a particularantigen (an “antigen-specific response”). In one embodiment, an immuneresponse is a T cell response, such as a CD4+ response or a CD8+response. In another embodiment, the response is a B cell response, andresults in the production of specific antibodies.

In some cases, the method of making a mutant multifunctional Cas9protein (e.g., having DNA binding activity and nuclease activity) ormutant nuclease-null Cas9 protein comprises identifying one or moreimmunodominant epitopes among full length amino acid sequences ofwild-type and/or modified Cas9 proteins, identifying a nucleic acidsequence for the one or more immunodominant epitopes, generating anucleic acid sequence for a target Cas9 protein which introduces one ormore mutations to disrupt the one or more immunodominant epitopes, andgenerating a mutant Cas9 protein from the generated nucleic acidsequence.

In certain embodiments, the mutations alter one or more amino acidresidues in the amino acid sequence of a target Cas protein. In somecases, the mutation replaces a leucine (L) residue at position 241 witha glycine (G), as numbered relative to the position numbering of aStreptococcus pyogenes Cas9 amino acid sequence (SEQ ID NO:1). A Cas9protein variant comprising L241G is identified herein as Cas9-α2 (SEQ IDNO:2). In other cases, the mutation replaces a leucine (L) residue atposition 616 with a glycine (G) as numbered relative to the positionnumbering of a Streptococcus pyogenes Cas9 protein. A Cas9 proteinvariant comprising a L241G mutation is identified herein as Cas9-β2 (SEQID NO:3). In some cases, mutations are generated at both positions, suchthat the Cas9 protein variant comprises amino acid substitutions at oneor both amino acid positions L241 and L616, as numbered relative to theposition numbering of a Streptococcus pyogenes Cas9 protein. A Cas9protein variant comprising L241G and L616G mutations is identifiedherein as Cas9-α2-β2 (SEQ ID NO:4). Preferably, the Cas9 variant proteinretains its wild-type enzymatic activity (e.g., nuclease, nickase,DNA-binding activity). In some cases, the mutant/modified Cas9 proteinis encoded by the amino acid sequence of SEQ ID NO:2. In other cases,the mutant/modified Cas9 protein is encoded by the amino acid sequenceof SEQ ID NO:3 or SEQ ID NO:4. Preferably, the mutant Cas9 proteinretains DNA binding and nuclease activity but is less likely to triggeran adverse immune response in a subject having pre-existing immunity toCas9.

In some cases, the wild type Cas protein is Streptococcus pyogenes Cas9(SpCas9). In other cases, the wild type Cas protein is a Cas9 orthologfrom another bacterial species such as, for example, Staphylococcusaureus and Streptococcus thermophiles. SpCas9 is the most extensivelystudied Cas9 protein but other orthologs are being explored to overcomesome of the limitations of SpCas9. These include the large size thatleaves little space for packaging additional sequences.

Linear immunogenic epitopes of a Cas9 protein, a modified Cas9 protein,or any portion of the Cas protein to be applied in a CRISPR/Cas systemare identified using, without limitation, publicly available algorithms(e.g., Immune Epitope Database and Analysis Resource (IEDB)). Predictedpeptides are ranked according to their immunogenicity score and will besynthesized to be used to detect the T cell response.

As used herein, the term “CRISPR/Cas” (Clustered Regularly InterspacedPalindromic Repeats/CRISPR associated) refers to a targeted genomeediting system that harnesses sequence-specific nuclease activity of aCas protein. CRISPR systems belong to different classes, with differentrepeat patterns, sets of genes, and species ranges.

Immunodominant epitopes can be identified and silenced to generate aCRISPR/Cas tool that induces minimal host immune response. In somecases, identified epitopes are modified by mutation or deletion of thenecessary amino acids to abolish MHC binding, or by mutation or deletionof the necessary amino acids to abolish TCR contact residues. In somecases, mutagenesis strategies employ the following steps: (a) designingCas gene fragments containing the desired mutations; (b) designing PCRprimers to amplify regions of a Cas gene that do not contain mutations;(c) performing a recombination-based cloning reaction (e.g., Golden Gateor variant thereof) or a BP reaction (i.e., using a BP clonase enzyme)in order to produce different Cas mutants (e.g., three different SpCas9mutants; single and double mutations); and (d) performing LR reaction(i.e., using a LR clonase enzyme) to clone the mutated Cas gene in aplasmid containing a promoter and Poly A to be able to express the gene.For review of the recombination based cloning systems, see Festa et al.,Proteomics. 2013; 13(9):1381-1399.

To assess Cas9 functionality and off-target effects after modification,nuclease activity of genetically modified Cas9 variants is measured.Cells are transfected with Cas9 mutants and gRNA targeting endogenous orexogenous genes, and on-target CRISPR/Cas9 mutations in cultured cellscaused by nuclease function of Cas9 are identified using deep sequencingand/or a surveyor nuclease assay. In other cases, Cas9 targetrecognition and binding function is achieved by transfecting cells withModified Cas9s and gRNA targeting endogenous or exogenous genes andactivation/repression mediators (e.g., SAM, VP64, p65AD, VPR, KRAB) andmeasuring the expression of genes. In some cases, off-target activity ofWT and modified Cas9 proteins are assessed using next generationsequencing.

Due to natural exposure, pre-existing immunity directed against thevector and less commonly the transgene is a common challenge in genetherapy. Being a bacterial protein, SpCas9 is likely to trigger cellularand humoral immune reaction in humans, as was demonstrated in naïvemice. More alarmingly, the ubiquity of S. pyogenes with 700 millioninfections annually, suggests that pre-existing immunity to SpCas9 inhealthy individuals is a reasonable concern. Accordingly, in anotheraspect, this disclosure provides a method for screening human patientsto identify patients more likely to have an adverse immune response togene therapy using a CRISPR/Cas9 system. In this manner, human patientscan be classified as good or poor candidates to receiveCRISPR/Cas9-based gene therapy based on the likelihood of the subjecthaving an adverse reaction to one or more components of the CRISPR/Cas9system. For example, candidate patients can be screened for pre-existingimmunity to Cas9. In some cases, pre-existing Cas9 immunity is apredictive biomarker of toxicity or adverse response toCRISPR/Cas9-based gene therapy.

Upon identification of a human patient having pre-existing immunity toCas9, a genetically modified Cas9 having reduced immunogenicity asdescribed herein could be used in place of an unmodified Cas9 for thatpatient's CRISPR/Cas9-based gene therapy. In some cases, Cas9immunogenicity in such a patient can also be limited or reduced byco-expressing molecules associated with immune evasion including, butnot limited to, PD-L1, CTLA-4, IL-10, IDO-1, antisense HLA class I, andβ₂M.

In order to identify immunogenic epitopes that can potentially beremoved or mutated, peripheral blood mononuclear cells (PBMCs) arecollected from individuals who have been infected with specificpathogens, individuals with known autoimmune disorders, healthyindividuals, etc. and are exposed to (i) Cas9 protein or any specificfragments of it; (ii) a modified Cas9 protein or any specific fragmentof a modified Cas9 protein; or (iii) antigen presenting cells (APCs)expressing Cas9, modified Cas9, a fragment of Cas9, a fragment ofmodified Cas9, or cells targeted with CRISPR/Cas9 system (in any form)expressing Cas9, modified Cas9, a fragment of Cas9, or a fragment ofmodified Cas9.

As used herein, the term “antigen presenting cells” or “APCs” refers tocells of the immune system used for presenting antigen to T cells. APCsinclude dendritic cells, monocytes, macrophages, marginal zone Kupffercells, microglia, Langerhans cells, T cells, and B cells. Preferably,the APCs are an individual's own APCs (meaning, derived or obtained fromthe individual) that can be briefly cultured and transduced with geneticmaterial (e.g., lentiviral clones) ex vivo. This is a preferred methodbecause it would enable the clinical study of an individual's own T cellrepertoire. In some cases, the APCs are an established cell line thatacts as an APC and has a human leukocyte antigen (HLA) type that matchesthe T cells. With established cell lines, it may be possible to maintaina population of cells already programmed with a wide variety of antigensthat can be used in repeated experiments. In other cases, the APCs arean established APC cell line that displays a highly common HLA type; anestablished APC cell line that is programmed to display an HLA type thatmatches the T cells; an established APC cell line that has beenengineered to produce a detectable marker protein (e.g., eGFP, mCherry,luciferase, etc.) upon induction by an activated T cell, an establishedAPC cell line that has been engineered to produce any other detectablesignal when induced by an activated T cell; or APCs into which areporter gene construct is introduced simultaneously with the cDNA. Thisreporter gene construct would be triggered to signal if the APC isinduced to mature after T cell activation.

APCs comprising particular gene constructs can be obtained by cDNAdelivery. Preferably, cDNA is introduced into a cell in a form thatsupports protein expression. The cDNA could be a gene encoding Cas9 fromany organism, a library encoding fragments of Cas9 protein that includepotentially immunogenic peptides (to map specific epitopes), a geneencoding a modified version of Cas9, or a sequence encoding fragments ofCas9 or Modified Cas9 to investigate which epitope(s) induces a T cellresponse. cDNA can be introduced into the cells using any appropriatemethod including, without limitation, lentivirus transduction,retrovirus transduction, other viral delivery systems, electroporationor nucleoporation, delivery of RNA, chemical transfection, or deliveryof an exogenous protein or proteins traceable to the library.

To perform the screening method, a population of T cells (e.g.,experimentally produced cells or cells from individuals who have beeninfected with specific pathogens, individuals with known autoimmunedisorders, healthy individuals, etc.) is mixed with a portion of Cas9protein as an antigen. Activated T cells will be measured as anindication of epitope recognition by T cells (effector T cells,cytotoxic T cells, helper T cells, memory T cells, natural killer Tcells, or regulatory T cells). T cell activation can be measured usingany appropriate method including, without limitation, methods formeasuring T cell proliferation (e.g., limiting dilutions culture);cytokine secretion (e.g., ELISPOT, intracellular staining); cytokinecapture (e.g. Miltenyi Biotec commercial IFN-γ secretion assay);tetramer (or any MHC-multimer) staining; spectratyping and biosensorassays to detect specific CDR3 of T cell populations of interest; andimmunophenotyping of activated T cells (e.g., CD25, CD69, CD137, CD107).

In another aspect, provided herein is a method of identifying andtreating a subject at risk of having a Cas9 antigen-specific CD8+ T cellimmune response. In some cases, the method comprises (a) detecting oneor more immunodominant Cas9 epitopes in a biological sample obtainedfrom the subject, wherein the detection of the one or moreimmunodominant Cas9 epitopes identifies the subject as havingpre-existing immunity to Cas9; (b) treating the subject identified in(a) with CRISPR/Cas9-based gene therapy, wherein treating comprisesintroducing into a cell from the identified subject an engineered,non-naturally occurring Type II CRISPR-Cas system comprising amultifunctional Cas9 protein and at least one guide RNA that targets andhybridizes to a target sequence of a DNA molecule in a cell, wherein theDNA molecule encodes and the cell expresses at least one gene product,and wherein the Cas9 protein comprises one or more mutations selectedfrom the group consisting of L241G and L616G (including double mutantL241G/L616G) with reference to the position numbering of a Streptococcuspyogenes Cas9 protein (SEQ ID NO:1), whereby expression of the at leastone gene product is altered and a disease associated with the geneproduct is treated. Exemplary immunodominant Cas9 epitopes are shown inTable 3. As used herein, the term “identifying” refers to any action orset of actions that allows a clinician to recognize a subject as one whomay benefit from the methods and compositions provided herein.Preferably, the identified subject is one who is in need of atolerogenic antigen-specific (e.g., Cas9-specific) immune response priorto or during CRISPR/Cas-based gene therapy as provided herein.

As used herein, the term “antigen-specific” refers to any immuneresponse that results from the presence of the antigen, or portionthereof, or that generates molecules that specifically recognize or bindthe antigen. For example, where the immune response is antigen-specificantibody production, antibodies are produced that specifically bind theantigen. As another example, where the immune response isantigen-specific CD8+ T cell proliferation and/or activity, theproliferation and/or activity can result from recognition of theantigen, or portion thereof, alone or in complex with MEW molecules. Insome cases, the antigen-specific immune response is a Cas9-specificimmune response.

For the methods provided herein, foreign nucleic acids (i.e., thosewhich are not part of a cell's natural nucleic acid composition) may beintroduced into a cell using any method known to those skilled in theart for such introduction. Such methods include transfection,transduction, viral transduction, microinjection, lipofection,nucleofection, nanoparticle bombardment, transformation, conjugation andthe like. One of skill in the art will readily understand and adapt suchmethods using readily identifiable literature sources. As used herein,the term “undesired immune response” refers to any undesired immuneresponse that results from exposure to an antigen, promotes orexacerbates a disease, disorder or condition provided herein (or asymptom thereof), or is symptomatic of a disease, disorder or conditionprovided herein. Such immune responses generally have a negative impacton a subject's health or is symptomatic of a negative impact on asubject's health. Undesired immune responses include Cas9antigen-specific CD8+ T cell proliferation and/or activity. Desiredimmune responses, therefore, include the inhibition in the stimulationor activation of CD8+ T cells, the inhibition of CD8+ T cellproliferation, the inhibition of the production of cytokines by CD8+ Tcells, etc. Methods for testing these immune responses are providedherein or are otherwise known to those of ordinary skill in the art.

In another aspect, provided herein is a method of reducing an undesiredCas9-specific CD8+ T cell immune response in a subject who will receiveCRISPR/Cas9-based gene therapy. In certain embodiments, the methodcomprises introducing into a cell from a subject identified as havingpre-existing immunity to Cas9 an engineered, programmable, non-naturallyoccurring Type II CRISPR-Cas system comprising a multifunctional Cas9protein and at least one guide RNA that targets and hybridizes to atarget sequence of a DNA molecule in a cell, wherein the DNA moleculeencodes and the cell expresses at least one gene product, and whereinthe Cas9 protein comprises one or more amino acid substitutions selectedfrom the group consisting of L241G, L616G, and L241G/L616G as numberedrelative to SEQ ID NO:1, whereby expression of the at least one geneproduct is altered and whereby a Cas9-specific CD8+ T cell immuneresponse is reduced relative to that produced by a cell comprising anaturally occurring Cas9 protein, a synthetic wild-type Cas9 protein, oran engineered non-naturally occurring Type II CRISPR/Cas system whereinthe Cas9 protein does not comprise the mutation. The introducing stepcan be performed ex vivo or in vivo.

In a further aspect, provided herein is a method of generating a variantCas9 protein, where the Cas9 variant is less immunogenic when expressedin a human cell yet retains its DNA binding/targeting capacity and itscapacity for transcriptional activation or repression. For example, avariant Cas9 generated according to a method described herein retainsits capacity to modulate transcription of endogenous genes and reportergenes (e.g., TTN, MIAT genes). In certain embodiments, a variant Cas9protein made by a method provided herein is less immunogenic relative toa non-variant Cas9 protein when expressed in a human cell yet retainsits DNA cleavage activity. In some cases, the method comprises using apolynucleotide mutagenesis procedure to generate a population of mutantsof the Cas9 polynucleotide shown in SEQ ID NO:5, wherein the populationof mutant Cas9 polynucleotides encodes Cas9 polypeptide variants havingat least one amino acid substitution selected from the group consistingof L241G and L616G as numbered relative to SEQ ID NO:1; and expressing apopulation of Cas9 polypeptide variants encoded by the population ofCas9 polynucleotide mutants; so that a variant of a Cas9 polypeptideshown in SEQ ID NO:1 is made.

In some cases, the method further comprises screening members of apopulation of Cas9 polypeptide variants so as to identify a variant thatexhibits a decreased immunogenicity when expressed in a human cell ascompared to the Cas9 polypeptide shown in SEQ ID NO:1 but retainscleavage and/or binding activity relative to the activity of a Cas9polypeptide without the at least one amino acid substitution.

Illustrative methods of mutagenesis protocols are shown, for example, inthe following Examples. In addition, a wide variety of techniques forgenerating variant polynucleotides and polypeptides have been well knownin the art for many years, for example site-directed mutagenesis (see,e.g. Carter et al., 1986, Nucl. Acids Res. 13:4331; Zoller et al., 1987,Nucl. Acids Res. 10:6487),

In another aspect, provided herein is a variant Cas9 protein made by amethod provided herein. Typically, the substitution variant exhibits oneor more altered properties as compared to the Cas9 polypeptide shown inSEQ ID NO:1, for example, a decreased immunogenicity.

Embodiments of this disclosure also include polynucleotides encoding theCas9 variants disclosed herein, for example an isolated polynucleotidehaving at least a 90%-100% sequence identity to a polynucleotideencoding a variant Cas9 polypeptide as disclosed herein. In some cases,a polynucleotide encoding a variant Cas9 polypeptide as disclosed hereinis in a vector. In some cases, the vector is in a host cell (e.g., abacterial cell, a human cell, or other eukaryotic cell).

A nucleic acid sequence encoding the desired variant Cas9 polypeptideonce isolated or synthesized, can be cloned into any suitable expressionvector using convenient restriction sites. Expression vectors usuallyinclude an origin of replication, a promoter, a translation initiationsite, optionally a signal peptide, a polyadenylation site, and atranscription termination site. These vectors also usually contain anantibiotic marker gene for selection. Suitable expression vectors may beplasmids, cosmids, or viruses including retroviruses. The codingsequence for the polypeptide is placed under the control of anappropriate promoter, control elements and a transcriptional terminatorso that the DNA sequence encoding the polypeptide is transcribed intoRNA in the host cell transformed by the expression vector construct. Thecoding sequence may or may not contain a signal peptide or leadersequence for secretion of the polypeptide out of the host cell. Numerousexpression vectors and systems are known, both for prokaryotes andeukaryotes, and the selection of an appropriate system is a matter ofchoice. Expression and purification of the polyprotein product of theinvention can be easily performed by one skilled in the art. See,Sambrook et al., “Molecular cloning-A Laboratory Manual, secondedition.”

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995). As appropriate, procedures involving the use ofcommercially available kits and reagents are generally carried out inaccordance with manufacturer defined protocols and/or parameters unlessotherwise noted.

So that the methods and compositions provided herein may more readily beunderstood, certain terms are defined:

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Any reference to “or” herein is intended toencompass “and/or” unless otherwise stated.

The terms “comprising”, “comprises” and “comprised of as used herein aresynonymous with “including”, “includes” or “containing”, “contains”, andare inclusive or open-ended and do not exclude additional, non-recitedmembers, elements, or method steps. The phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” “having,” “containing,”“involving,” and variations thereof, is meant to encompass the itemslisted thereafter and additional items. Use of ordinal terms such as“first,” “second,” “third,” etc., in the claims to modify a claimelement does not by itself connote any priority, precedence, or order ofone claim element over another or the temporal order in which acts of amethod are performed. Ordinal terms are used merely as labels todistinguish one claim element having a certain name from another elementhaving a same name (but for use of the ordinal term), to distinguish theclaim elements.

The terms “nucleic acid” and “nucleic acid molecule,” as used herein,refer to a compound comprising a nucleobase and an acidic moiety, e.g.,a nucleoside, a nucleotide, or a polymer of nucleotides. Typically,polymeric nucleic acids, e.g., nucleic acid molecules comprising threeor more nucleotides are linear molecules, in which adjacent nucleotidesare linked to each other via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g. nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to an oligonucleotide chain comprising three or moreindividual nucleotide residues. As used herein, the terms“oligonucleotide” and “polynucleotide” can be used interchangeably torefer to a polymer of nucleotides (e.g., a string of at least threenucleotides). In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA. Nucleic acids may benaturally occurring, for example, in the context of a genome, atranscript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid,chromosome, chromatid, or other naturally occurring nucleic acidmolecule. On the other hand, a nucleic acid molecule may be anon-naturally occurring molecule, e.g., a recombinant DNA or RNA, anartificial chromosome, an engineered genome, or fragment thereof, or asynthetic DNA, RNA, DNA/RNA hybrid, or include non-naturally occurringnucleotides or nucleosides. Furthermore, the terms “nucleic acid,”“DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e.analogs having other than a phosphodiester backbone. Nucleic acids canbe purified from natural sources, produced using recombinant expressionsystems and optionally purified, chemically synthesized, etc. Whereappropriate, e.g., in the case of chemically synthesized molecules,nucleic acids can comprise nucleoside analogs such as analogs havingchemically modified bases or sugars, and backbone modifications. Anucleic acid sequence is presented in the 5′ to 3′ direction unlessotherwise indicated. In some embodiments, a nucleic acid is or comprisesnatural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine,uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadeno sine, 7-deazaadenosine,7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine,and 2-thiocytidine); chemically modified bases; biologically modifiedbases (e.g., methylated bases); intercalated bases; modified sugars(e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose);and/or modified phosphate groups (e.g., phosphorothioates and5′-N-phosphoramidite linkages).

The terms “protein,” “peptide,” and “polypeptide” are usedinterchangeably herein and refer to a polymer of amino acid residueslinked together by peptide (amide) bonds. The terms refer to a protein,peptide, or polypeptide of any size, structure, or function. Typically,a protein, peptide, or polypeptide will be at least three amino acidslong. A protein, peptide, or polypeptide may refer to an individualprotein or a collection of proteins. One or more of the amino acids in aprotein, peptide, or polypeptide may be modified, for example, by theaddition of a chemical entity such as a carbohydrate group, a hydroxylgroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. A protein, peptide, or polypeptide may also be asingle molecule or may be a multi-molecular complex. A protein, peptide,or polypeptide may be just a fragment of a naturally occurring proteinor peptide. A protein, peptide, or polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination thereof. Aprotein may comprise different domains, for example, a nucleic acidbinding domain and a nucleic acid cleavage domain. In some embodiments,a protein comprises a proteinaceous part, e.g., an amino acid sequenceconstituting a nucleic acid binding domain, and an organic compound,e.g., a compound that can act as a nucleic acid cleavage agent.

As used herein, “modifying” (“modify”) one or more target nucleic acidsequences refers to changing all or a portion of a (one or more) targetnucleic acid sequence and includes the cleavage, introduction(insertion), replacement, and/or deletion (removal) of all or a portionof a target nucleic acid sequence. All or a portion of a target nucleicacid sequence can be completely or partially modified using the methodsprovided herein. For example, modifying a target nucleic acid sequenceincludes replacing all or a portion of a target nucleic acid sequencewith one or more nucleotides (e.g., an exogenous nucleic acid sequence)or removing or deleting all or a portion (e.g., one or more nucleotides)of a target nucleic acid sequence. Modifying the one or more targetnucleic acid sequences also includes introducing or inserting one ormore nucleotides (e.g., an exogenous sequence) into (within) one or moretarget nucleic acid sequences.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same, and inthe same relative position, when comparing the two sequences. Sequencesimilarity can be determined in a number of different manners. Todetermine sequence identity, sequences can be aligned using the methodsand computer programs, including alignment algorithms such as BLAST(available on the World Wide Web at ncbi.nlm.nih.gov/BLAST) and FASTA(available in the Genetics Computing Group (GCG) package).

As used herein, a “coding sequence” can be a sequence which “encodes” aparticular gene, such as a Cas9 gene, for example. A coding sequence isa nucleic acid sequence which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A transcription termination sequence will usually be located3′ to the coding sequence.

As used herein, the term “operably linked” refers to an arrangement ofelements wherein the components so described are configured so as toperform their usual function. Thus, control sequences (e.g., promotersequences, ribosome binding sites, polyadenylation signals,transcription termination sequences, upstream regulatory domains,enhancers, and the like) operably linked to a coding sequence arecapable of effecting the expression of the coding sequence. The controlsequences need not be contiguous with the coding sequence, so long asthey function to direct the expression thereof.

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions are included tobetter appreciate the teaching of the present invention.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein by reference.

EXAMPLES

Presented in this example are experiments demonstrating thatimmunodominant Cas9 epitopes can be mutated to reduce immunogenicity inhuman cells without loss of Cas9 DNA binding or nuclease activity.

Materials & Methods

Detection of Cas9-Specific Serum Antibodies in Healthy Controls: Healthycontrol sera (n=183) used in this study, and previously described(Anderson et al., Oral oncology 51:662-667 (2015)), are a subset of amolecular epidemiology study of head and neck cancer at the MD AndersonCancer Center, collected between January 2006 and September 2008. S.pyogenes lysate was prepared by sonication of bacterial pellets fromovernight cultures of S. pyogenes ATCC 19615 in the presence of 1 pillof complete Protease Inhibitor (Sigma-Aldrich) after 3 cycles offreezing and thawing. Serum antibody detection was performed usingELISA. 96-well plates were coated with 20 μg/mL of recombinant S.pyogenes Cas9 nuclease (New England Biolabs, Ipswich, Mass.) or S.pyogenes lysate. Sera were diluted 1:50 in 10% E. coli lysate preparedin 5% milk-PBST (0.2% tween) (Wang et al., Proteomics Clin Appl7:378-383 (2013)), incubated with shaking for 2 hours at roomtemperature, and added to the specified wells in duplicate. Horseradishperoxidase (HRP) anti-human IgG Abs (Jackson ImmunoResearchLaboratories, West Grove, Pa.) were added at 1:10,000, and detectedusing Supersignal ELISA Femto Chemiluminescent substrate (Thermo FisherScientific, Waltham, Mass.). Luminescence was detected as relative lightunits (RLU) on a Glomax 96 Microplate Luminometer (Promega, Madison,Wis.) at 425 nm. To establish cut-off values, a RLU ratio >(the mean+3standard deviations) of 125 randomly chosen control samples wasdesignated positive (FIG. 2, dotted and dashed lines for bacteriallysate and Cas9 protein, respectively).

Cas9 candidate T cell epitope prediction: Previously describedprediction strategies were used to predict candidate Cas9 T cellepitopes. Briefly, we predicted MHC class I restricted 9-mer and 10-mercandidate epitopes derived from the Cas9 protein (Uniprot—Q99ZW2) forHLA A*02:01. The protein reference sequence was entered into 5 differentprediction algorithms; 3 MHC-binding: IEDB-consensus binding (Moutaftsiet al., Nature biotechnology 24:817 (2006)), NetMHCpan binding (Hoof etal., Immunogenetics 61:1 (2009)), Syfpeithi (Rammensee et al.,Immunogenetics 50:213-219 (1999)), and 2 antigen-processing algorithms:IEDBconsensus processing, ANN processing (Tenzer et al., Cellular andMolecular Life Sciences 62, 1025-1037 (2005)). The individual scoresfrom each of the prediction algorithms were then normalized within thepool of predicted peptides after exclusion of poor binders as previouslydetailed (Chowell et al., Proc Natl Acad Sci USA 112:E1754-1762 (2015);Krishna & Anderson, Vaccine Design: Methods and Protocols: Volume 1:Vaccines for Human Diseases, 779-796 (2016)), and the average normalizedbinding scores were used to rerank the candidate peptides. The top 38candidate peptides (Table 3) were selected for experimental testing. TheIEDB consensus MHC-binding prediction algorithm (available at iedb.orgon the World Wide Web) was applied to obtain a list of high binding Cas9peptides, each of which was assigned a normalized binding score (Sb).The immunogenicity score (Si) was calculated for each peptide based onits amino acid hydrophobicity (ANN-Hydro) (Chowell, D. et al., Proc NatlAcad Sci USA 112:E1754-1762 (2015)).

Ex vivo stimulation and epitope mapping of Cas9 by ELISpot: Allperipheral blood mononuclear cells (PBMCs) were obtained from healthyindividuals with written informed consent under ASU's InstitutionalReview Board. PBMCs were isolated from fresh heparinized blood byFicoll-Hypaque (GE Healthcare, UK) density gradient centrifugation andstimulated as previously described (Krishna & Anderson, Vaccine Design:Methods and Protocols: Volume 1: Vaccines for Human Diseases, 779-796(2016)). Briefly, predicted Cas9 peptides with Sb<0.148 (N=38) weresynthesized (>80% purity) by Proimmune, UK. Each peptide wasreconstituted at 1 mg/mL in sterile PBS and pools were created by mixing3-4 candidate peptides. Sterile multiscreen ELISpot plates (MerckMillipore, Billerica, Mass., USA) were coated overnight with 5 μg/wellof anti-IFN-γ capture antibody (clone D1K, Mabtech, USA) diluted insterile PBS. Frozen PBMCs were thawed rapidly and recombinant human IL-2(20 U/mL, R&D Systems) was added. They were then stimulated intriplicates with 10 μg/mL Cas9 peptide pools (or individual peptides),pre-mixed CEF pool as a positive control (ProImmune, UK), or DMSO as anegative control in the anti-IFN-γ-coated ELISpot plates, (MerckMillipore, Billerica, Mass., USA) and incubated in a 37° C., 5% CO₂incubator for 48 hours. Plates were washed three times for 5 min eachwith ELISpot buffer (PBS +0.5% FBS) and incubated with 1 μg/mLanti-IFN-γ secondary detection antibody (clone 7-B6-1, Mabtech, USA) for2 hrs at room temperature, washed and incubated with 1 μg/mLStreptavidin ALP conjugate for 1 hour at room temperature. The wellswere washed again with ELISpot buffer and spots were developed byincubating for 8-10 min with detection buffer (33 μL NBT, 16.5 μL BCIP,in 100 mM Tris-HCl pH 9, 1 mM MgCl₂, 150 mM NaCl). Plates were left todry for 2 days and spots were read using the AID ELISpot reader(Autoimmun Diagnostika GmbH, Germany). The average number of spotforming units for each triplicate was calculated for each test peptideor peptide pool and subtracted from the background signal.

Autologous APC generation from healthy individual PBMCs: AutologousCD40L-activated B cell APCs were generated from healthy donors byincubating whole PBMCs with irradiated (32 Gy) K562-cell line expressinghuman CD40L (KCD40L) at a ratio of 4:1 (800,000 PBMCs to 200,000irradiated KCD40Ls) in each well. The cells were maintained in B cellmedia (BCM) consisting of IMDM (Gibco, USA), 10% heat-inactivated humanserum (Gemini Bio Products, CA, USA), and Antibiotic-Antimycotic(Anti-Anti, Gibco, USA). BCM was supplemented with 10 ng/mL recombinanthuman IL-4 (R&D Systems, MN, USA), 2 μg/mL Cyclosporin A (Sigma-Aldrich,CA, USA), and insulin transferrin supplement (ITES, Lonza, Md., USA).APCs were re-stimulated with fresh irradiated KCD40Ls on days 5 and 10,after washing with PBS and expanding into a whole 24-well plate. Aftertwo weeks, APC purity was assessed by CD19+CD86+ expressing cells usingflow cytometry, and were used for T cell stimulation after >90% purity.APCs were either restimulated up to 4 weeks or cryopreserved forre-expansion as necessary.

T cell stimulation by autologous APCs: Antigen-specific T cells weregenerated by stimulating healthy donor B cell APCs by peptide pulsing ofspecific Cas9 epitopes. Peptide pulsing of APCs was done under BCM 5%human serum, with recombinant IL-4. Twenty-four hours later, on day 1,APCs were washed and incubated with thawed whole PBMCs at a ratio of 1:2(200,000 APCs: 400,000 PBMCs) in a 24-well plate in BCM supplementedwith 20 U/mL recombinant human IL-2 (R&D Systems, MN, USA) and 5 ng/mLIL-7 (R&D Systems, MN, USA). On day 5, partial media exchange wasperformed by replacing half the well with fresh BCM and IL-2. On day 10,fresh APCs were peptide pulsed in a new 24-well plate. On day 11,expanded T cells were restimulated with peptide-pulsed APCs similar today 1. T cells were used for T cell assays or immunophenotyped after day18.

Flow cytometry staining for T cells: Cells were washed once in MACSbuffer (containing PBS, 1% BSA, 0.5 mM EDTA), centrifuged at 550 g for 5min and re-suspended in 200 μL MACS buffer. Cells were stained in 100 μLof staining buffer containing anti-CD137, conjugated with phycoerythrin(PE, clone 4B4-1; BD Biosciences, USA), anti-CD8-PC5 (clone B9.11;Beckman Coulter 1:100), anti-CD4 (clone SK3; BioLegend, 1:200),anti-CD14 (clone 63D3; BioLegend, 1:200), and anti-CD19 (clone HIB19;BioLegend, 1:200), all conjugated to Fluorescein isothiocyanate (FITC)for exclusion gates, for 30 min on ice. Samples were covered andincubated for 30 min on ice, washed twice in PBS, and resuspended in 1mL PBS prior to analysis. Measurements were performed using an AttuneAcoustic Focusing Cytometer. Lymphocytes were first identified byforward (FSC) and side scatter (SSC) gating. CD4/CD14/CD19/CD56-negativecells (bin gate) were selected, and activated Pentamer+(or CD137+) Tcells were identified within the CD8+ gate. Analysis was performed usingAttune Cytometric Software V2.1.

Pentamer staining for T cell immunophenotyping: The followingHLA-A*02:01 PE-conjugated Cas9 pentamers were obtained from ProImmune:F2A-D-CUS-A*02:01-ILEDIVLTL-Pentamer,F2A-D-CUS-A*02:01-NLIALSLGL-Pentamer, 007-influenza A MP8-66-GILGFVFTL-Pentamer. T cells were washed twice in MACS buffer with5% human serum and centrifuged at 550 g for 5 min each time. They werethen re-suspended in 100 μL staining buffer (MACS buffer, with 5% humanserum and 1 mM Dasatanib (ThermoFisher Scientific, MA, USA). Each of thepentamers was added to resuspended T cells, stimulated with therespective peptide or APCs at a concentration of 1:100. Samples wereincubated at room temperature for 30 min in the dark, then washed twicein MACS buffer. Cells were stained in 100 μL MACS buffer withanti-CD8-PC5, anti-CD4-FITC, anti-CD14-FITC, and anti-CD19-FITC forexclusion gates, Samples were then washed twice with PBS and analyzed byflow cytometry. For flow cytometric analysis, all samples were acquiredwith Attune flow cytometer (ThermoFisher Scientific, MA, USA) andanalyzed using the Attune software. Gates for expression of differentmarkers and pentamers were determined based on flow minus one (FMO)samples for each color after doublet discrimination. Percentages fromeach of the gated populations were used for the analysis.

Vector Design and Construction:

Modified Cas9 plasmids—Human codon-optimized Streptococcus pyogenes Cas9sequence was amplified from pSpCas9 (pX330; Addgene plasmid ID: 42230),using forward and reverse primers and inserted within gateway entryvectors using golden gate reaction. Desired mutations were designedwithin gBlocks (Integrated DNA Technologies). The gblocks and ampliconswere then cloned into entry vectors using golden gate reaction. All theprimers and gblocks sequences are listed in supplementary notes. Next,the Cas9 vectors and CAG promoter cassettes were cloned into anappropriate gateway destination vector via LR reaction (Invitrogen).

U6-sgRNA-MS2 plasmids—These plasmids were constructed by insertingeither 14-bp or 20-bp spacers of gRNAs into sgRNA (MS2) cloning backbone(Addgene plasmid ID: 61424) at BbsI site. gRNA sequences are listed inTable 1.

TABLE 1 Sequences of gRNAs MIAT-14bp gRNA GAGGCTGAGCGCAC (SEQ ID NO: 9)TTN-14bp gRNA GGAAGTCTCCTTTG (SEQ ID NO: 10) Reporter2-20bp GTCCCCTCCACCCCACAGTG (SEQ ID NO: 11) gRNA CR10-14bp-gRNAGCATCAGGAACATGT (SEQ ID NO: 12) EMX1-20bp gRNACACC GAGTCCGAGCAGAAGAAGAA  (SEQ ID NO: 13)

Cell culture for endogenous target mutation and activation: HEK293FTcell line was purchased from ATCC and maintained in Dulbecco's modifiedEagle's medium (DMEM—Life Technologies) containing 10% fetal bovineserum (FBS—Life Technologies), 2 mM glutamine, 1.0 mM sodium pyruvate(Life Technologies) and 1% penicillinstreptomycin (Life Technologies) inincubators at 37° C. and 5% CO₂. Polyethylenimine (PEI) was used totransfect HEK293FT cells seeded into 24-well plates. Transfectioncomplexes were prepared according to manufacturer's instructions.

Flow cytometry for Quantifying Cas9 Function: HEK293FT cells wereco-transfected with 10 ng gRNA, 200 ng Cas9 constructs, 100 ng reporterplasmid and 25 ng EBFP2 expressing plasmid as the transfection control.Flow cytometry data were collected 48 h after transfection. Cells weretrypsinized and centrifuged at 500 g for 5 min at 4° C. The supernatantwas then removed, and the cells were resuspended in Hank's Balanced SaltSolution without calcium or magnesium supplemented with 2.5% FBS. BDCelesta was used to obtain flow cytometry measurements in synthetic genecircuits with the following settings: EBFP, measured with a 405 nm laserand a 407/421 filter; EYFP, measured with a 488 nm laser and a 490/515filter; iRFP, measured with a 640 nm laser and a 50/785 filter. At least200,000 events were gathered from each sample. Flow cytometry data wasanalyzed using FlowJo software. Briefly, cells expressing more than>2×10² A.U of EBFP (transfection marker) were gated after gating thecells based on FSC and SSC (to exclude debris) and the geometric mean ofEYFP was calculated. A sample was excluded if there were less than 300events in the gated population. A representative flow cytometry gatingis depicted in FIG. 11B.

Quantitative RT-PCR Analysis: HEK293FT cells were co-transfected with 10ng gRNA, 200 ng Cas9 constructs, 100 ng MS2-P65-HSF1 (Addgene plasmidID: 61423) and 25 ng transfection control. Cells were lysed, and RNA wasextracted using RNeasy Plus mini kit (Qiagen) 72 hours posttransfection, followed by cDNA synthesis using the High-CapacityRNA-to-cDNA Kit (Thermo fisher). qRT-PCR was performed using SYBR GreenPCR Master Mix (Thermo fisher) using a QuantStudio 3 by AppliedBiosystems. All analyses were normalized to 18s rRNA (ΔCt) andfold-changes were calculated against un-transfected controls (2−ΔΔCt).Primer sequences for qPCR are listed in Table 2.

TABLE 2 Sequences of primers Cas9 fragment 1-FWttttGGTCTCTAGGTCCACCATGGACTATAAGGACCACGA (SEQ ID NO: 14)Cas9 fragment1-RV tttggtctcaGAACAGCTGGTTGTAGGTCTGCA (SEQ ID NO: 15)Cas9 fragment2-FW ttttGGTCTCTACCAACCGGAAAGTGACCGTGAAG (SEQ ID NO: 16)Cas9 fragment2-RV ttttGGTCTCAAAGCTTACTTTTTCTTTTTTGCC (SEQ ID NO: 17)qPCRMIAT-FW TGGCTGGGGTTTGAACCTTT (SEQ ID NO: 18) qPCR-MIAT RVAGGAAGCTGTTCCAGACTGC (SEQ ID NO: 19) qPCRTTN FWTGTTGCCACTGGTGCTAAAG (SEQ ID NO: 20) qPCR-TTN-RVACAGCAGTCTTCTCCGCTTC (SEQ ID NO: 21) PCR-EMX1-FWCCATCCCCTTCTGTGAATGT (SEQ ID NO: 22) PCR-EMX1-RVGGAGATTGGAGACACGGAGA (SEQ ID NO: 23)

Endogenous Indel Analysis: HEK293FT cells were co-transfected with 200ng of Cas9 plasmids, 10 ng of gRNA coding cassette and 25 ngtransfection control. 72 hours later, transfected cells were dissociatedand spun down at 200 g for 5 minutes at room temperature. Genomic DNAwas extracted using 50 μl of QuickExtract DNA extraction solution(Epicentre) according to the manufacturer's instructions. Genomic DNAwas amplified by PCR using primers flanking the targeted region,Illumina Tru-Seq library was created by ligating partial adaptors and aunique barcode to the DNA samples. Next, a small number of PCR cycleswere performed to complete the partial adaptors. Equal amounts of eachsample were then pooled and sequenced on Illumina Tru-Seq platform with2×150 run parameters, which yielded approximately 80,000 reads persample. Sequencing was performed using a 2×150 paired-end (PE)configuration by CCIB DNA Core Facility at Massachusetts GeneralHospital (Cambridge, Mass., USA). The reads were aligned to the targetgene reference in Mus musculus genome using Geneious software, 9-1-5. Todetect the indels (insertions and deletions of nucleic acid sequence atthe site of double-strand break), each mutation was evaluated carefullyin order to exclude the ones that are caused by sequencing error or anyoff-target mutation. The variant frequencies (percentage to total)assigned to each read containing indels were summed up. i.e., indelpercentage=total number of indel containing reads/total number of reads.The minimum number of analyzed reads per sample was 70,000.

RNA Sequencing for Quantifying Activator Specificity: HEK293FT cellswere co-transfected with 10 ng gRNA for MIAT locus, 200 ng Cas9constructs, 100 ng MS2-P65-HSF1 (Addgene plasmid ID: 61423) and 25 ngtransfection control. Total RNA was extracted 72 hours post transfectionusing RNeasy Plus mini kit (Qiagen) and sent to UCLA TCGB core on dryice. Ribosomal RNA depletion, and single read library preparation wereperformed at UCLA core followed by RNA sequencing using NextSeq500.Coverage was 14 million reads per sample. FASTQ files with single-ended75 bp reads were then aligned to the human GRCh38 reference genomesequence (Ensembl release 90) with STAR 54, and uniquely-mapped readcounts (an average of 14.8 million reads per sample) were obtained withCufflink (Trapnell et al., Nature Protocols 7:562-578 (2012)). The readcounts for each sample were then normalized for the library size to CPM(counts per million reads) with edgeR (Robinson et al., Bioinformatics26:139-140 (2010)). Custom R scripts were then used to generate plots.

Results

Detection of Cas9-Specific Serum Antibodies in Healthy Controls: Theinventors first investigated whether healthy donors, in particular thosewith previous exposure to Streptococcus pyogenes, have detectable IgGantibodies (Abs) to Streptococcus pyogenes Cas9 (SpCas9). Of 143 healthycontrol sera screened, 49.0% had detectable Abs against S. pyogeneslysate as detected using ELISA (FIG. 2). This positive subset (closedcircles) along with 12 of the sera negative for S. pyogenes lysate thathad the highest RLU value were screened for Abs against syntheticwild-type Cas9. At least 21.0% (n=30) of healthy individuals in thisstudy had Cas9-specific Abs (FIG. 2), confirming pre-existing immuneresponse to Cas9.

Next, HLA-A*02:01-restricted T cell epitopes derived from SpCas9 werepredicted using a model based on both HLA binding and biochemicalproperties of immunogenicity (Table 3; the top 5 are shown in FIG. 3).Using the inventors' previously reported prediction model (Chowell, D.,et al., Proc Natl Acad Sci USA, 2015. 112(14):E1754-62), based on a WICclass I binding probability and a peptide immunogenicity probability,Cas9 peptides with predicted high binding and high immunogenicity wereprioritized. In brief, IEDB consensus WIC-binding prediction algorithm(available at iedb.org on the World Wide Web) was applied to obtain alist of high binding Cas9 peptides for HLA-A*02:01, each of which wasassigned a normalized binding score (S_(b)). Next, an immunogenicityscore (S_(i)) was calculated for each peptide based on its amino acidhydrophobicity. According to this prediction model, peptides with lowS_(b) (high binders) and high S_(i) (more hydrophobic) are expected tobe more immunogenic. The calculated normalized binding (S_(b)) andimmunogenicity (S_(i)) scores for each peptide (FIG. 3) were plotted topredict the more immunogenic epitopes, which are expected to have bothhigh HLA binding (low S_(b)) and more hydrophobicity (high S_(i)).

Identification of two Cas9 immunodominant T cell epitopes: To determinewhether healthy donor peripheral blood mononuclear cells (PBMCs) hadmeasurable T cell reactivity against predicted Cas9 MHC class Iepitopes, the top 38 peptides (Table 1) were synthetized and groupedinto 10 pools (each containing 3-4 peptides) for screening memory T cellresponse in healthy individuals using ELISpot. Peptide-specific T cellimmunity was measured using IFN-γ secretion ELISpot assays with PBMCsderived from 12 healthy individuals, and immunoreactive epitopes wereidentified within pools 3 or 5 in 83.0% of the donors tested (FIG. 4).The seven individual peptides from pools 3 and 5 were evaluated by IFN-γELISpot and the dominant immunogenic epitopes were SpCas9_293-301 andSpCas9_668-676, designated peptides α and β, from pools 3 and 5,respectively. The position of peptides α and β within the proteinstructure is shown in FIG. 5. The individual peptides within pools thatwere positive for any donor were evaluated for this donor by IFN-γELISpot. The immunoreactivity and position of the 38 predicted peptides(a few of which are overlapping) within the Cas9 protein are shown inFIG. 6.

Peptides α and β are shown as red dots on the epitope prediction plotand their sequences and predicted ranking are shown in FIG. 3 and Table3. As predicted, these peptides had low Sb and high Si values. Both theimmunodominant (α and β) and subdominant (γ and δ) T cell epitopesidentified by IFN-γ ELISpot were within the top 5 epitopes predicted byour previously described immunogenicity model. Sequence similarity ofpeptides α (previously identified by inventors as immunodominant epitope85) and β (previously identified by inventors as immunodominant epitope94) to amino acid sequences in known proteins was investigated usingProtein BLAST and the IEDB epitope database. A peptide was considered‘similar’ to α or β if at least 7 of 9 (78%) amino acid residues (thatare not the second or ninth) were matching. None of these two peptidesresembled known epitopes in the IEDB database, but similarity to otherCas9 orthologs and other bacterial proteins was detected (Tables 4 and5). Epitope β has sequence similarity to a peptide derived from theNeisseria meningitidis peptide chain release factor 2 protein (ILEDIVLTL(SEQ ID NO:28) versus ILEGIVLTL (SEQ ID NO:72)).

TABLE 4Sequence homology of epitope a to amino acid sequences from known proteinsEpitope Sequence SEQ ID NO: Similarity (%) Protein Sequence ID Source 1NLIALSLGL 27 9/9 (100%) type II CRISPR RNA-guided WP 014612333.1Streptococcus endonuclease Cas9 dysgalactioe 2 NLIALSLGL 27 9/9 (100%)type II CRISPR RNA-guided WP 054279288.1 Streptococcus endonuclease Cas9phocoe 3 NLIALSLGL 27 9/9 (100%) type II CRISPR RNA-guidedWP 067062573.1 Streptococcus endonuclease Cas9 pantholopis 4 NLIALSLGL27 9/9 (100%) type II CRISPR RNA-guided WP 048800889.1 Streptococcusendonuclease Cas9 constellatus 5 NLIALSLGL 27 9/9 (100%)type II CRISPR RNA-guided WP 002304487.1 Streptococcus endonuclease Cas9mutans 6 NLIALSLGL 27 9/9 (100%) type II CRISPR RNA-guidedWP 049516684.1 Streptococcus endonuclease Cas9 anginosus 7 NLIALSLGL 279/9 (100%) type II CRISPR RNA-guided WP 003079701.1 Streptococcusendonuclease Cas9 macacae 8 NLIALSLGL 27 9/9 (100%)type II CRISPR RNA-guided GAD40915.1 Streptococcus endonuclease Cas9intermedius SK54 9 NLIA F SLGL 62 8/9 (89%) Full = RNA polymerase-Q6LV34.1 Photobacterium associated profundum protein RapA; AltName: SS9Full = ATP-dependent helicase HepA 10 NLI S LSLGL 63 8/9 (89%)type II CRISPR RNA-guided WP 096633625.1 Streptococcus endonuclease Cas9parauberis 11 NLIAL A LGL 64 8/9 (89%) type II CRISPR RNA-guidedWP 075103982.1 Streptococcus endonuclease Cas9 cuniculi 12 NLIAL A LGL64 8/9 (89%) type II CRISPR RNA-guided WP 058692367.1 Streptococcusendonuclease Cas9 gallolyticus 13 NLIAL A LGL 64 8/9 (89%)type II CRISPR RNA-guided WP 061100419.1 Streptococcus endonuclease Cas9pasteurianus 14 NLIAL A LGL 64 8/9 (89%) type II CRISPR RNA-guidedWP 018363470.1 Streptococcus endonuclease Cas9 caballi 15 NLIAL A LGL 648/9 (89%) type II CRISPR RNA-guided WP 099412266.1 Streptococcusendonuclease Cas9 macedonicus 16 NLIAL A LGL 64 8/9 (89%)type II CRISPR RNA-guided WP 014334983.1 Streptococcus endonuclease Cas9infontarius 17 DLIAL Y LGL 65 7/9 (78%) Full = NADH-quinone A8I421.1Azorhizobium oxidoreductase caulinodons subunit N; AltName: ORS 571Full = NADH dehydrogenase I subunit N; AltName: Full = NDH-1 subunit N18 NLLALALGL 66 7/9 (78%) type II CRISPR RNA-guided WP 007896501.1Streptococcus endonuclease Cas9 pseudoporcinus 19 NLI G LA LGL 677/9 (78%) type II CRISPR RNA-guided WP 061587801.1 Streptococcusendonuclease Cas9 oralis 20 NL V AL A LGL 68 7/9 (78%)type II CRISPR RNA-guided WP 074862269.1 Streptococcus endonuclease Cas9equinus 21 NL V AL V LGL 69 7/9 (78%) type II CRISPR RNA-guidedWP 020917064.1 Streptococcus endonuclease Cas9 lutetiensis 22 S LIA FSLGL 70 7/9 (78%) ectoine/hydroxyectoine WP 086160327.1 Streptomyces ABCsp. SCSIO transporter permease 03032 subunit EhuD 23 Y LIAL A LGL 717/9 (78%) ectoine/hydroxyectoine WP 026413155.1 Actinomadura ABColigospora transporter permease subunit EhuD

TABLE 5Sequence homology of epitope β to amino acid sequences from known proteinsEpitope Sequence SEQ ID NO: Similarity (%) Protein Sequence ID Source 1ILEDIVLTL 28 9/9 (100%) type II CRISPR RNA-guided WP 084916602.1Streptococcus endonuclease Cas9 dysgalactiae 2 ILEDIVLTL 28 9/9 (100%)type II CRISPR RNA-guided WP 074484960.1 Streptococcus endonuclease Cas9henryi 3 ILEDIVLTL 28 9/9 (100%) type II CRISPR RNA-guidedWP 003088697.1 Streptococcus endonuclease Cas9 ratti 4 ILEDIVLTL 289/9 (100%) type II CRISPR RNA-guided WP 044681799.1 Streptococcusendonuclease Cas9 suis 5 ILEDIVLTL 28 9/9 (100%)type II CRISPR RNA-guided WP 024786433.1 Streptococcus endonuclease Cas9mutans 6 ILEDIVLTL 28 9/9 (100%) type II CRISPR RNA-guidedWP 057491067.1 Streptococcus endonuclease Cas9 orisasini 7 ILEDIVLTL 289/9 (100%) type II CRISPR RNA-guided WP 082312238.1 Streptococcusendonuclease Cas9 intermedius 8 ILE G IVLTL 72 8/9 (89%)peptide chain release factor NP 275123.1 Neisseria 2 meningitidis MC58 9ILEDIV Q TL 73 8/9 (89%) type II CRISPR RNA-guided EAO61901.1Streptococcus endonuclease Cas9 agalactiae 10 ILEDIV Q TL 73 8/9 (89%)type II CRISPR RNA-guided WP 070454905.1 Streptococcus endonuclease Cas9sp. HMSC063D10 11 V LEDIVLTL 74 8/9 (89%) type II CRISPR RNA-guidedWP 075346866.1 Streptococcus endonuclease Cas9 sp. ‘covioe’ 12 V LEDIVLS L 75 7/9 (78%) type II CRISPR RNA-guided WP 093650272.1 Streptococcusendonuclease Cas9 yarani 13 ILE N IV H TL 76 7/9 (78%)type II CRISPR RNA-guided KYF37509.1 Streptococcus endonuclease Cas9mitis 14 ILE N IV H TL 76 7/9 (78%) type II CRISPR RNA-guidedWP 084972088.1 Streptococcus endonuclease Cas9 oralis 15 ILE N IV H TL76 7/9 (78%) type II CRISPR RNA-guided WP 045635197.1 Streptococcusendonuclease Cas9 gordonii

Next, the inventors focused on the immunodominant epitope β.Antigen-specific T cells were expanded for 18 days in vitro bycoculturing healthy donor PBMCs with peptide β-pulsed autologous antigenpresenting cells (APCs). Cas9-specific CD8+ T cell responses wereassessed by flow cytometry. CD8+ T cells specific for the HLA-A*0201/βpentamer were detected after stimulation (3.09%; FIG. 7A).

It was hypothesized that mutation of the MHC-binding anchor residues ofthe identified immunogenic epitopes would abolish specific T cellrecognition (FIG. 7A). The epitope anchor residues (2nd and 9th) are notonly necessary for peptide binding to the MHC groove, but are alsocrucial for recognition by the T cell receptor 36. The percentage ofCD8+β pentamer+ T cells decreased to 0.3% when APCs were pulsed with themutated peptide (β2; FIG. 7B) compared with 3.09% with the wild typepeptide (β; FIG. 7A). The reactivity of healthy donor T cells tomodified peptides α or β with mutations in residues 2, 9, or both(sequences are shown in FIG. 7C) was measured using IFN-γ ELISpot assay.The epitope-specific T cell reactivity was markedly reduced with themutant peptides (FIG. 7D, FIG. 8). The average reduction was 8 fold fromα to α29 and 25 fold from β to β29 (n=12; p<0.047; FIG. 8).

A modified Cas9 construct was produced by mutating the second residue ofpeptide (L616G; Cas9-β2) and tested the function of this new Cas9variant in comparison with wild type Cas9 (WT-Cas9) in the context ofDNA cleavage and transcriptional modulation. To examine the nucleaseactivity of Cas9-β2 and compare with WT-Cas9, Cas9-β2 or WT-Cas9 weretargeted to an endogenous locus (FMX-1) and measured percent indelformation (FIGS. 7E, 7F). Our data demonstrate that Cas9-β2 retainsnuclease capacity in the locus we studied as well as on a syntheticpromoter (FIG. 7F, FIG. 9A). Next, it was determined whether Cas9-β2 cansuccessfully recognize and bind its target DNA leading totranscriptional modulation. First, it was tested in the context ofenhanced transgene expression from a synthetic CRISPR responsivepromoter in HEK293 cells using 14 nt gRNAs and aptamer mediatedrecruitment of transcriptional modulators similar to what we had shownbefore (FIG. 9B). Having shown successful transgene activation, it wasinvestigated whether this variant retains such capacity within thechromosomal contexts of endogenous genes. Cells were transfected withplasmids encoding Cas9-β2 or WT-Cas9 and 14 nt gRNAs against twodifferent endogenous genes (TTN and MIAT). qRT-PCR analysis showed thatthis variant successfully led to target gene expression (FIGS. 7G-7I).To further characterize Cas9-β2 specificity, genome-wide RNA sequencingwas performed after targeting Cas9-β2 or WT-Cas9 to the MIAT locus fortranscriptional activation. The results demonstrated no significantincrease in undesired off-target activity by Cas9-β2 as compared toWT-Cas9 (FIG. 7J).

To show the extensibility of our approach, another Cas9 variant wasgenerated by mutating the second residue of peptide α (L241G; Cas9-α2).Cas9-α2 also demonstrated DNA cleavage and transcriptional modulationfunctionality comparable with WT-Cas9 (FIGS. 10A-10E). When T cells werestimulated with APCs spiked with peptide α2, the percentage ofCD8+CD137+ T cells (a marker of T cell activation) was decreased by 2.3fold as compared to WT peptide α stimulation (FIG. 10F).

The detection of pre-existing B cell and T cell immunity to the mostwidely used nuclease ortholog of the CRISPR/Cas9 tool in a significantproportion of healthy humans confirms previous studies in mice and shedslight on the need for more studies of the immunological risks of thissystem. The CD8+ T cell immunity we observed is likely memory responses,as they are observed without ex vivo stimulation. Following 18 days of Tcell stimulation by peptides α or β, expansion of naive T cells is notprecluded. This suggests that the expression of Cas9 in naiveindividuals may trigger a T cell response that could prevent subsequentadministration. This could be avoided by switching to Cas9 orthologsfrom other bacterial species, but attention needs to be given toindividual and distinct immune repertoires. This can be difficult giventhe epitope conservation across Cas9 proteins from multipleStreptococcus species and resemblance to sequences from other bacterialproteins such as the common pathogen N. meningitidis thatasymptomatically colonizes the nasopharynx in 10% of the population.Therefore, selective deimmunization (also known as immunosilencing) ofCas9 can represent an attractive alternative. Selective deimmunizationcan be an effective alternative for CRISPR applications in patientswhere systemic immunosuppression proves to be difficult, such as inpatients with chronic infectious diseases. This strategy can beimportant particularly when longer expression of Cas9 will be desiredfor epigenetic therapy.

Conventional methods of deimmunizing non-human therapeutic proteins relyon trial-and-error mutagenesis, machine learning, and often includesdeletion of whole regions of the protein. Here, as a general principle,it was determined that alteration of one of the anchor residues of animmunodominant epitope abolished specific T cell recognition. However,HLA allotype diversity and the existence of numerous epitopes in thelarge Cas9 protein complicate the process of complete deimmunization.The overall impact of removal of select immunodominant epitopes remainsto be seen; similar approaches for other proteins have resulted inreduction and enhancement of the immunogenicity of subdominant epitopes.Non-specific immune suppressive approaches may complement thesestrategies for complete deimmunization. One attractive strategy is theco-expression of Cas9 with gRNAs targeting immune modulatory moleculessuch as programmed death-ligand 1 (PD-L1) or Indoleamine 2,3-Dioxygenase1 (IDO1) to further boost immunosilencing. It is believed thatdeimmunized Cas9 will be useful for therapeutic CRISPR applications as abetter understanding of the immunological consequences of this systemdevelops.

The mutated Cas9 protein sequences are as follows, with peptides 85 (α)(SEQ ID NO:27) and 94 (β) (SEQ ID NO:28) in bold, underlined text:

Cas9-α2: (SEQ ID NO: 2)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKK NGLFGNGIALSLGL TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Cas9-β2  (SEQ ID NO: 3)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKK NGLFGNLIALSLGL TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED IGEDIVLTL TLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Cas9-α2-β2  (SEQ ID NO: 4)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKK NGLFGNGIALSLGL TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED IGEDIVLTLTL FEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Cas9-β2:  (SEQ ID NO: 6)ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGA TAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAG TACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAA GGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTG ATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCA GAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGAT GGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGA AGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCC ACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA TCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCG ACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAA AACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAG ACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTTA TTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAA CTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCA GTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAG AGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACC ACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATT TTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTT CTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGA ACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCAC CTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCG GGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAA ACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGA AGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACC TGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAG CTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGA AAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGA GGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCA ACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAAT GAGGAAAACGAGGACATTGGTGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGAT GATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGC GGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCA GTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGC GATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCA GACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTG ATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATG AAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAA ACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTG GACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTT TCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGC GACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACG CCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGA ACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTG GCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGT GAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGT GCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCC TGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTG CGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAG CAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTC TGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTG CGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCT TCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTG GGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCA AAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCAT GGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTG AAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAG AATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGA ACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAA CAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTC CAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGG ATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCC CCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGT GCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC AGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGT  AA Cas9-α2:  (SEQ ID NO: 7) ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGA TAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAG TACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAA GGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTG ATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCA GAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGAT GGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGA AGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCC ACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA TCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCG ACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAA AACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAG ACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACGGTA TTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAA CTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCA GTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAG AGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACC ACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATT TTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTT CTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGA ACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCAC CTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCG GGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAA ACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGA AGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACC TGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAG CTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGA AAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGA GGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCA ACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAAT GAGGAAAACGAGGACATTCTTGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGAT GATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGC GGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCA GTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGC GATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCA GACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTG ATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATG AAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAA ACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTG GACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTT TCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGC GACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACG CCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGA ACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTG GCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGT GAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGT GCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCC TGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTG CGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAG CAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTC TGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTG CGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCT TCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTG GGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCA AAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCAT GGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTG AAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAG AATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGA ACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAA CAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTC CAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGG ATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCC CCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGT GCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC AGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGT  AAG Cas9-α2-β2  (SEQ ID NO: 8)ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGA TAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAG TACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAA GGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTG ATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCA GAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGAT GGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGA AGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCC ACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTA TCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCG ACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAA AACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAG ACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACGGTA TTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAA CTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCA GTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAG AGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACC ACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATT TTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTT CTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGA ACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCAC CTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCG GGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAA ACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGA AGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACC TGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAG CTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGA AAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGA GGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCA ACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAAT GAGGAAAACGAGGACATTGGTGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGAT GATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGC GGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCA GTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGC TGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGC GATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCA GACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTG ATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATG AAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAA ACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTG GACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTT TCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGC GACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACG CCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGA ACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTG GCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGT GAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGT GCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCC TGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTG CGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAG CAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTC TGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTG CGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCT TCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTG GGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCA AAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCAT GGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTG AAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAG AATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGA ACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAA CAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTC CAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGG ATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCC CCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGT GCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTC AGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGT 

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. A method of reducing anundesired Cas9-specific CD8+ T cell immune response in a subject whowill receive CRISPR/Cas9-based gene therapy, the method comprising:introducing into a cell from a subject identified as having pre-existingimmunity to Cas9 an engineered, programmable, non-naturally occurringType II CRISPR-Cas system comprising a multifunctional Cas9 protein andat least one guide RNA that targets and hybridizes to a target sequenceof a DNA molecule in a cell, wherein the DNA molecule encodes and thecell expresses at least one gene product, and wherein the Cas9 proteincomprises a mutation selected from the group consisting of L616G andL241G/L616G as numbered relative to SEQ ID NO:1, whereby expression ofthe at least one gene product is altered and whereby a Cas9-specificCD8+ T cell immune response is reduced relative to that produced by acell comprising an engineered, programmable, non-naturally occurringType II CRISPR-Cas system wherein the Cas9 protein does not comprise themutation.
 5. The method of claim 4, wherein the Cas9 protein comprisinga mutation has an amino acid sequence selected from SEQ ID NO:3 and SEQID NO:4.
 6. The method of claim 4, wherein the introducing step isperformed ex vivo or in vivo.
 8. A variant Cas9 protein having the aminoacid sequence of SEQ ID NO:3.
 9. A variant Cas9 protein having the aminoacid sequence of SEQ ID NO:4.
 10. An isolated polynucleotide encoding avariant Cas9 polypeptide of claim
 8. 11. A vector comprising thepolynucleotide of claim
 10. 12. A host cell comprising the vector ofclaim
 11. 13. A method of making a variant Cas9 protein, the methodcomprising: introducing at least one mutation into the Cas9polynucleotide of SEQ ID NO:5, wherein the mutated Cas9 polynucleotideencodes a variant Cas9 protein having at least one amino acidsubstitution selected from the group consisting of L241G and L616G asnumbered relative to SEQ ID NO:1; and expressing the variant Cas9protein.
 14. The method of claim 13, wherein the variant Cas9 proteinexhibits decreased immunogenicity as compared to the Cas9 protein of SEQID NO:1 but retains the cleavage and binding activity of the Cas9protein of SEQ ID NO:
 1. 15. A variant Cas9 protein made by the methodof claim
 13. 16. An isolated polynucleotide encoding a variant Cas9polypeptide of claim
 9. 17. A vector comprising the polynucleotide ofclaim
 16. 18. A host cell comprising the vector of claim 17.